Seasonal Chemical Evaluation of Miconia chamissois Naudin from Brazilian Savanna

Miconia chamissois Naudin is a species from the Cerrado, which is being increasingly researched for its therapeutic potential. The aim of this study was to obtain a standardized extract and to evaluate seasonal chemical variations. Seven batches of aqueous extracts from leaves were produced for the standardization. These extracts were evaluated for total solids, polyphenol (TPC) and flavonoid content (TFC), vitexin derivative content, antioxidant activity; thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC) profiles were generated. For the seasonal study, leaves were collected from five different periods (May 2017 to August 2018). The results were correlated with meteorological data (global radiation, temperature, and rainfall index). Using chromatographic and spectroscopic techniques, apigenin C-glycosides (vitexin/isovitexin) and derivatives, luteolin C-glycosides (orientin/isoorientin) and derivatives, a quercetin glycoside, miconioside B, matteucinol-7-O-β-apiofuranosyl (1 → 6) -β-glucopyranoside, and farrerol were identified. Quality parameters, including chemical marker quantification by HPLC, and biological activity, are described. In the extract standardization process, all the evaluated parameters showed low variability. The seasonality study revealed no significant correlations (p < 0.05) between TPC or TFC content and meteorological data. These results showed that it is possible to obtain extracts from M. chamissois at any time of the year without significant differences in composition.


Introduction
Cerrado, with a predominantly dry and hot climate, is recognized as the richest savanna in the world, home to 11,627 species of native plants already cataloged [1]. Cerrado comprises several phytophysiognomies and has a prevalence of some botanical families with commercial, cultural, and social importance (food, ethnobotany, religious, and others), as well as the potential for ecological restoration [2].
The richness of the Cerrado is such that the range and potential of bioactive compounds produced by Cerrado species can be considered greater than those of the Amazon Forest, representing an interesting field of investigation with medicinal plants and conservation of natural resources [3].
Melastomataceae is the sixth most abundant botanical family of angiosperms in Brazil, comprising more than 1300 species, with Miconia, Leandra, Tibouchina, Microlicia, and Clidemia among the most diverse genera. In all of Brazil, 267 species from the Miconia genus have been described and are distributed in these phytogeographic domains: Amazon, mill to obtain a powder with 30 mesh (0.59 mm) using a sieve integrated into the mill. This powder was extracted in water by infusion at 70 • C to 50 • C at a ratio of 1:10. The aqueous extract was then lyophilized (VirTis SP Scientific Advantage Plus XL-70 Benchtop Freeze Dryer) and stored at −20 • C.
The yield of aqueous extract from Miconia chamissois Naudin (AEMC) was calculated as the weight percentage of the dried powdered leaves.
The total solids content was determined using an infrared moisture detector (Gehaka ® model IV2000, São Paulo, Brazil) from 2 mL of the sample. The analysis was performed in triplicate, and the results are expressed as the weight percentage of the dried leaf material.

Extraction Process Standardization
To evaluate the standardization and reproducibility of the extraction process, seven equal batches of aqueous extracts from leaves were prepared (B1-B7). Leaves collected in May 2017 at Lago do Cedro, Brasília, Federal District, Brazil (coordinates 15 • 53 48.0" S, 47 • 56 36.1" W) were used to prepare the seven batches. Total solids, polyphenol and flavonoid contents, TLC and HPLC profiles, vitexin derivate content, and antioxidant activity were evaluated from these batches.

Polyphenol and Flavonoid Contents
Total polyphenol (TPC) and total flavonoid (TFC) contents in AEMCs were determined using a modified colorimetric method proposed by Kumazawa et al. (2004) [19]. The TPC assay was performed in a 96-well plate, with 50 µL of standard or sample, 50 µL of 10% calcium carbonate (Na 2 CO 3 ), and 50 µL of 1N Folin reagent. One hour after the addition of Folin reagent to the reaction medium, absorbance was measured at 760 nm using a Perkin Elmer EnSpire plate reader. The results were expressed in µg equivalents of gallic acid (µg EGA/mg or percentage of total polyphenol content (%)). The TFC assay was performed in a 96-well plate by adding 100 µL of 2% aluminum chloride (AlCl 3 ) solubilized in 40% ethanol and 100 µL of standard/sample. One hour after the addition of 2% aluminum chloride (AlCl 3 ) to the reaction medium, absorbance was measured at 420 nm using a Perkin Elmer EnSpire plate reader. The results were expressed in µg equivalents of quercetin per mg of extract (µg QE/mg) or percentage of total flavonoid content (%). The analyses were performed in triplicate.
Vitexin equivalents (VE) were determined with a standard curve generated using vitexin in the range of 8 to 100 µg/mL. The sample was analyzed at a concentration of 5 mg/mL in a methanol and water solution (6:4). The data from the standard curve were used to estimate the vitexin equivalents in the sample using linear regression of the data. Analyses were performed for all seven batches.

UHPLC-MS/MS Assay
UHPLC analyses were performed using a Waters Acquity H-series UPLC coupled to a Waters Acquity PDA detector in series with a Xevo triple quadrupole mass spectrometer. A Waters Acquity UPLC BEH C18 column (1.7 µm, 2.1 mm × 100 mm) was used, with mobile phases A = 0.1% formic acid and B = acetonitrile. The column was held at 35 • C with a flow rate of 0.35 mL/min, with 100% A and 0% B with a linear gradient of 40% A and 60% B at 30 min, followed by 4 min re-equilibration to the original conditions. The PDA was monitored continuously over a range of 230-500 nm. The injection volume was 15 µL. The mass spectrometer was operated in several different modes for separate injections. Initially, positive ion full scan positive ion electrospray 'survey scans' were acquired over the range m/z 100 to 1500 every 0.3 s, with a cone voltage of 30 V. Scanwave daughter scans at 10 V and 20 V collision energy (CE) at 2000 m/z per second were automatically acquired from the strongest ions. To unequivocally determine molecular weights, full negative ion electrospray spectra were subsequently acquired from m/z 100 to 1000 every 0.4 s using a cone voltage ramp, followed by targeted MS/MS scans with a cone voltage of 30 V and collision energy of 40 V from the relevant major [M − H] − ions. The ion source temperature was 130 • C, the desolvation gas was nitrogen at 950 L/h, the desolvation temperature was 450 • C, and the capillary voltage was 2.7 KV in all cases. For this assay, a mixture of seven batches of aqueous extracts from leaves was used.
2.5. Antioxidant Activity 2.5.1. DPPH Assay Antioxidant activity was evaluated by reducing the DPPH (2,2-Diphenyl-1-picrylhydrazyl) radical using an adapted methodology described by Blois (1958) [21]. Ascorbic acid was used as a positive control, and a standard curve was generated over the concentration range of 10 to 1000 µg/mL. Different AEMC samples were evaluated at a concentration of 3 µg/mL. The results were also expressed as inhibition percentage (%) and were determined using the following equation: AI (%) = 100 − (Sample Abs − Sample Blank) * 100/Control Abs AI = antioxidant inhibition (%) Sample Abs = sample absorbance Sample Blank = solvent Control Abs = inhibition

Phosphomolybdenum Method
Antioxidant activity was determined using the phosphomolybdenum method, as described by Pietro et al. (1999) [22]. Ascorbic acid was used as a positive control, and a standard curve was generated over the concentration range of 10-300 µg/mL. The AEMC was evaluated at a concentration of 125 µg/mL. The assay was performed by adding 1.0 mL of the reagent solution to 0.1 mL of the sample or the standard. The reagent solution consisted of 28 mM phosphate, 4 mM molybdate, and 0.6 M sulfuric acid. After a reaction time of 90 min in a water bath at 95 • C, the absorbance was measured at 695 nm using a Shimadzu ® UV-1800 (Software UVProve 2.33) spectrophotometer. The data obtained from the standard curve were used to estimate the equivalents of ascorbic acid content in the sample using linear regression. The development stage of the plants was observed, in which P1 was flowering/fruiting, P2 was fruiting, P3 was vegetative period, P4 was flowering/fruiting, and P5 was fruiting. The aqueous extract from these leaves was prepared by infusion following the method described above.
The meteorological data for 2017 and 2018 were provided by the Automatic Agrometeorological Station of Agroclimatology Laboratory from the University of Brasília. The data were limited to Lago do Cedro, Brasília, Federal District, Brazil, and included meteorological parameters, such as global radiation (MJm 2 d −1 ), maximum temperature ( • C), minimum temperature ( • C), and rainfall index (mm) from the period of 17 January to 18 August.
Meteorological data were correlated with total solids content, TPC and TFC contents, vitexin derivate content, and antioxidant activity by DPPH assay. Pearson's linear correlation coefficient (r) was used to determine the correlation level.

Statistical Analysis
Microsoft Office Excel ® 2016 software and GraphPad Prism ® Version 5.01 were used for statistical analysis. The results are expressed as the average plus standard deviation and relative deviation standard. ANOVA tests followed by Kruskal-Wallis or Dunn's multiple comparison tests were used in different assays. Pearson's correlation was used for the seasonal study, using linear correlations evaluated according to Callegari-Jacques considering |r| = 0-0.3 (weak), |r| = 0.3-0.6 (moderate), and |r| = 0.6-0.9 (strong) [26].

Results and Discussion
Herbal medicines have a complex chemical constitution, and their pharmacological effect are often due to synergy between these compounds. Several factors can affect the chemical composition of a plant extracts, such as growth, harvest, drying, the extraction process, and storage conditions [27]. To secure a constant composition of herbal preparations, and consequently their efficacy and safety, it is necessary to ensure their pharmaceutical quality by standardizing the process for these products [20].
In this study, a method to obtain a standardized extract of M. chamissois Naudin was developed, and the seasonal chemical variability was evaluated.

Chemical Composition and Standardization
Seven AEMC batches were prepared according to the method described above. The reproducibility of the extraction process was evaluated in relation to total solid, polyphenol, and flavonoid contents, TLC and HPLC profiles, vitexin derivate content, and biological activity by antioxidant activity.
In the TLC analysis, the same chemical profile was observed for the seven batches, with five main spots. The retention factor (R f ) of these spots was 0.17, 0.33, 0.43, 0.54, and 0.61 ( Figure 1).
Herbal medicines have a complex chemical constitution, and their pharmacological effect are often due to synergy between these compounds. Several factors can affect the chemical composition of a plant extracts, such as growth, harvest, drying, the extraction process, and storage conditions [27]. To secure a constant composition of herbal preparations, and consequently their efficacy and safety, it is necessary to ensure their pharmaceutical quality by standardizing the process for these products [20].
In this study, a method to obtain a standardized extract of M. chamissois Naudin was developed, and the seasonal chemical variability was evaluated.

Chemical Composition and Standardization
Seven AEMC batches were prepared according to the method described above. The reproducibility of the extraction process was evaluated in relation to total solid, polyphenol, and flavonoid contents, TLC and HPLC profiles, vitexin derivate content, and biological activity by antioxidant activity.
In the TLC analysis, the same chemical profile was observed for the seven batches, with five main spots. The retention factor (Rf) of these spots was 0.17, 0.33, 0.43, 0.54, and 0.61 ( Figure 1).  Pearson (r) linear correlation was performed to assess the correlation between total polyphenol content and extractable solids content. A weak negative correlation (r = −0.20; p = 0.37) was not significant between the evaluated parameters. In general, the weak correlation indicates that the parameters evaluated are unlikely to be comparable and associate; that is, there was no correlation.
Another Pearson (r) linear correlation was carried out to correlate the total flavonoid and total solids content. The results showed a strong negative non-significant correlation (r = −0.87; p = 0.02), which suggested a tendency of an inversely proportional relationship between the total flavonoid content and the total solids content.
Gontijo et al. (2019) found 3.56 ± 0.17 µg equivalent of rutin/mg of extract in aqueous extract of leaves of M. latecrenata (DC.) Naudin prepared by infusion (1:20) [28]. In M. albicans (Su.) Triana fruits 510.96 ± 8.64 mg QE 100 g −1 of total flavonoids was measured in July 2015, after a dry winter in the region where the fruits were collected and where the average annual temperature was 21.8 • C [29].
Compound detection by HPLC/DAD was performed by comparing the spectra obtained and retention times (t R ) of seven batches of AEMC and standards. The chromatogram profiles showed 12 peaks at 354 nm (Table 1 and Figure 2). The results in the Table 1 show the mean values of retention time and peak area. The chromatogram presented in Figure 2 is representative of the analysis of one of the batches (B4). In the identification of AEMC compounds by HPLC/DAD, the results showed similar UV spectra between peak 6 (t R 24.89 min) and vitexin (0.9959) and isovitexin (0.9951) standards (Figure 2A-C); the retention times were similar. This is in agreement with the data reported by Gomes et al. (2021) [11], who found a similarity between the peak at 24.8 min of the aqueous extract of M. chamissois Naudin leaves and vitexin (0.9968) and isovitexin (0.9963). Peak 6 was considered to be closely related to vitexin and isovitexin.
To quantify the compound corresponding to peak 6, a linear regression of the standard curve generated using vitexin as the standard (y = 104,860x − 109,129, r = 0.99) was used to determine the vitexin equivalent (VE) content. The data analysis of seven batches showed 13.67 ± 0.57 µg VE/mg of AEMC (RSD = 4.17%). In the identification of AEMC compounds by HPLC/DAD, the results showed similar UV spectra between peak 6 (tR 24.89 min) and vitexin (0.9959) and isovitexin (0.9951) standards (Figure 2A-C); the retention times were similar. This is in agreement with the data reported by Gomes et al. (2021) [11], who found a similarity between the peak at 24.8 min of the aqueous extract of M. chamissois Naudin leaves and vitexin (0.9968) and isovitexin (0.9963). Peak 6 was considered to be closely related to vitexin and isovitexin.
To quantify the compound corresponding to peak 6, a linear regression of the standard curve generated using vitexin as the standard (y = 104860x − 109129, r = 0.99) was used to determine the vitexin equivalent (VE) content. The data analysis of seven batches showed 13.67 ± 0.57 µg VE/mg of AEMC (RSD = 4.17%).
For the standardization process of AEMC, the biological activity by antioxidant activity using the DPPH assay was also evaluated. The seven batches of AEMC showed 59.34% ± 7.92 (RSD = 13.35%) of inhibition of the reduction of DPPH radical at a concentration of 3 µg/mL. The results of batches B1 to B7 showed significant differences; batch B1 was significantly different from B2 and B6, B1 from B5, B4 from B5, and B5 from B6 using ANOVA test of multiple comparisons (Dunn's) followed by the Kruskal-Wallis test.
Pearson correlation between antioxidant activity (DPPH assay) and total solids content or total polyphenol content, and flavonoid content was evaluated. The results revealed a weak positive correlation (r = 0.15; p = 0.42) between polyphenols and antioxidant activity and a moderate negative correlation between total flavonoids and antioxidant activity (r = −0.59; p = 0.14), with no significant difference (p < 0.05) between the chemical assays and DPPH antioxidant activity. There was a moderate positive correlation (r = 0.76; p = 0.06), but no significant correlation was found between antioxidant activity and total solids content.
Although the results demonstrated low variability in chemical composition, a larger variability in biological activity was observed among the seven batches tested. Nevertheless, considering the DPPH assay as a bioanalytical method, the precision should not exceed 15% (RSD) [38]. The variability in the antioxidant activity of the AEMC batches was within the allowed range.
Biological activity of several herbal medicinal products is due to the synergy between its many constituents [39]. Thus, evaluating the biological activity is an important tool for determining the quality of herbal products. Oxidative stress is present in the mechanism of many diseases, such as neurodegenerative [40], cardiovascular [41], and liver diseases [42], and in the aging process [43]. Therefore, antioxidant activity can be an important biological assay to ensure the efficacy and quality of herbal products.

UHPLC-MS/MS Analysis
To better characterize the chemical composition of AEMC, a UHPLC-MS/MS analysis was performed. Ten of the peaks detected matched the chromatographic profile obtained by HPLC/DAD, aiming to identify flavonoids ( Figure 3). The data obtained are listed in Table 2.    The identities of compounds present in AEMC were based on the results obtained compared with data in the literature or with similarity to compounds in the Metlin MS database, and it was not possible to confirm all of them due to the complexity of the sample, and the lack of reference MS data for some relatively obscure flavonoids. In particular, the reference MS data available for the mixed C and O glycosides of apigenin and luteolin are very limited.  [44,45]. In terms of the formal flavonoid glycoside fragmentation notation, the Z 1 ion was 429, and the subsequent 0.2 X 0 ion was 309. These glycosides are characterized by this intense ion at the mass of the aglycone + 23. The data strongly suggested presence of an O-hexosyl-C-hexosyl luteolin, such as compounds like 2"-O-glucosyl-8-C-glucosyl luteolin  [49,50]. These peaks together appear to correspond to peak 6 from the HPLC-UV/DAD analysis, identified as a vitexin/isovitexin derivative; we observed slightly better separation by UHPLC compared to that by HPLC.

Antioxidant Activity
To better characterize the antioxidant activity of AEMC, two additional methods were used: the phosphomolybdenum method and the lipid peroxidation assay.
For TBARS, AEMC did not inhibit the formation of reactive species. For the dried extract (50% ethanol v/v) of Miconia albicans leaves, an IC 50 value of 1338.34 µg/mL [36] was observed. Osbeckia parvifolia Arn. ethyl acetate extract of whole plants showed 59.6% TBARS inhibition [62]. Leaves of other species, such as Melastomastrum capitatum A. Fern. & A. Fern. showed TBARS inhibition of 86.86% ± 3.63 and 39.93% ± 1.07 for ethanol and aqueous extracts, respectively [63].  Table 3. The meteorological data are presented in Table 4, and the correlation results are presented in Table 5. The data represent the monthly average. According to the data obtained, there were no significant correlations (p < 0.05) between the TPC and TFC and meteorological data, with a strong negative correlation between global radiation, suggesting that the production of these metabolites is inversely proportional to the radiation index. Another strong negative correlation was observed between the antioxidant activity and rainfall index, suggesting that the antioxidant activity is inversely proportional to the rainfall index.

Seasonality Study
A moderate positive correlation between total solids and global radiation and a weak correlation between the other parameters were also observed.
Notably, the harvests were carried out in different reproductive periods, with P3 being the only harvest in which the species did not show flowering, fruiting or maturation; on 17 May, 17 November and 18 August, the highest total polyphenol content and lowest radiation were observed. Gobbo-Neto and Lopes (2007) understand the complexity of seasonality studies. In addition to meteorological and environmental parameters (water availability, radiation, temperature, soil, and altitude), the metabolic and hormonal conditions inherent in the development of the plant species must be attributed [15].
Interest in studies that assess changes in the secondary metabolism of plants has grown in recent decades and contributes to understanding the composition, adaptive capacity, and bioactive screening of species. When it comes to species from Cerrado, the interest is even greater because of the complexity and biological richness of this biome.
Studies of biological material from fauna and flora are very complex due to the object of the investigation itself, and the complexity increases when dealing with species from the Cerrado because of the diversity present in this biome. Zanatta et al. (2021) also pointed out that there may be a gap in the physiological response of plants. There will not necessarily be a positive correlation between composition and biological activity, especially with species from the Cerrado [64].
Melastomacatecae has adaptive capacity under different environmental conditions and, thus, can be present in all vegetation formations in the Cerrado [65]. Ishino et al. (2021) still emphasized the changes caused by anthropogenic issues and consequently provoked biological changes in the fauna and flora to guarantee protection. In a study of seasonality, some native plants may be stress-tolerant and may not show differences in physical and physical characteristics [66].

Conclusions
A standardized AEMC extract was obtained, and chemical marker quantification by HPLC was performed. Biological activity using the DPPH assay was proposed as an important quality parameter. In the extract's standardization process of the extract, all the parameters evaluated showed low variability. Moreover, AEMC showed high reactivity and potential antioxidant activity via the phosphomolybdenum complex. The chemical composition confirmed the presence of polyphenolic compounds already identified in the species, contributing to the chemical elucidation of the species that have been the object of study by various research groups. Using different chromatographic techniques, luteolin glycosides, apigenin glycosides, a quercetin glycoside, miconioside B, matteucinol-7-Oβ-apiofuranosyl (1 → 6)-β-glucopyranoside and farrerol were identified. Several of the main flavonoids were mixed C,O-diglycosides of apigenin and luteolin. The seasonal evaluation is of great value considering that there was no correlation between composition and activity versus meteorological parameters, indicating the temporal adaptability of the species. These results showed that it is possible to obtain extracts from M. chamissois at any time of the year without significant differences in composition.

Supplementary Materials:
The following are available online. Figure S1: UHPLC-UV/MS/MS data of peak 4; Figure S2: UHPLC-UV/MS/MS data of peak .5; Figure S3: UHPLC-UV/MS/MS data of peak 6; Figure S4: UHPLC-UV/MS/MS data of peak 7; Figure S5: UHPLC-UV/MS/MS data of peak 8; Figure S6: UHPLC-UV/MS/MS data of peak 12; Figure  Data Availability Statement: All data included in this study are available upon request by contact with the corresponding author.